Self and microbial glycosylceramides perform important immunological functions as NKT cell antigens presented by CD1d, a lipid-binding MHC-like molecule. These functions range from host defense against pathogens to the regulation of cancer rejection, autoimmunity and allergy. Further, synthetic glycosylceramides can be used as adjuvants of vaccines and can modulate, through the release of either Thl or Th2 cytokines, various disease conditions including diabetes, experimental allergic encephalomyelitis, lupus and cancer rejection. Other glycosylceramides elicit adaptive T cell responses that may be involved in Multiple Sclerosis. Thus, the program project uses glycosylceramides as a model antigen family to study glycolipid antigen presentation by CD1d molecules, with potential clinical applications, as well as fundamental issues in lipid transport and recognition. A multidisciplinary approach encompassing glycolipid analytic and synthetic chemistry, protein expression, biophysics and structural biology, as well as cellular immunology and cell biology, has been assembled to identify NKT ligands from natural sources and study fundamental aspects of their immunobiology. The past funding period has produced landmark accomplishments in identifying key self and microbial NKT cell antigens, characterizing lipid transfer proteins essential for their presentation, elucidating their crystal structures and developing a panel of synthetic variants to study their cell biology and their functional impact in vaccine and immunomodulation. These approaches will be pursued with the goal of developing a basic understanding of the cell biology of lipid uptake, trafficking and loading, of the biophysical and structural aspects of their binding to protein receptors such as CD1d, TCR and other lipid binding proteins;and integrating this knowledge in vivo at the functional level during NKT cell mediated immune responses to infection or to synthetic adjuvants. The individual research projects are as follows. Project 1. Savage: Identification and synthesis of microbial NKT ligands and design of structural variants and conjugates to understand their functional properties. Project 2. Teyton: Biochemical, biophysical and structural aspects of lipid interaction with CD1d, TCR and other lipid binding or transfer protein. Project 3. Bendelac: Functions associated with NKT ligand recognition in vivo in mice and intracellular trafficking properties of glycosylceramides PROJECT 1: TH1/TH2 Glycolipid Adjuvants (Savage, P) PROJECT 1 DESCRIPTION (provided by applicant): Natural killer T cells (NKT cells) play an important role in regulating immune responses. NKT cells are stimulated by glycolipid antigens presented by CD1d, and in the last few years significant advances have been made in understanding this process. Natural antigens for NKT cells have been identified, and crystal structures of CD1d bound to glycolipids have been solved. As factors leading to NKT cell stimulation are elucidated, the importance of glycolipid trafficking and specific interactions with T cell receptors (TCRs) is emerging. However, glycolipid trafficking and interactions with TCRs are not well understood. Proposed research includes development of labeled glycolipids for use in studying specific trafficking events;specifically, the influences of structural variations of glycolipids on trafficking and how differences in trafficking influence the Th1/Th2 bias of cytokine release by NKT cells. Studies of glycolipid and CD1d interactions with TCRs will involve incremental structural modifications of known antigens to determine requirements for association and stimulation. In addition, glycolipid functionality has bee identified that can be modified without impacting negatively NKT cell stimulation, and this information provides a means of using small molecules appended to glycolipids to modify the affinity of TCRs for glycolipid-CD1d complexes. It is anticipated that higher affinity will result in prolonged stimulation of NKT cells and increased cytokine release;however, the impact of this affinity on cytokine release profiles is not known and these studies will provide that information. The number of known organisms producing natural antigens for NKT cells is rather small, and bacteria related to those known to stimulate NKT cell directly will be screened for NKT stimulatory behavior. Structures of antigens will then be determined and confirmed through total synthesis. Relevance to Public Health: Responses of NKT cells influence disease states including infection, tumor rejection, and autoimmune diseases. An understanding of how glycolipids stimulate different responses from NKT cells will facilitate use of these responses to improve human health. Proposed research will increase this understanding while augmenting the arsenal of glycolipids that stimulate NKT cells.